[弓形虫免疫反应性多表位抗原在大肠杆菌中的可溶性表达及特性分析]

[Soluble expression and characterization of the immunoreactive multiepitope antigen of Toxoplasma gondii in E.coli].

作者信息

Yang Pei-liang, Chen Xiao-guang, Wang Yuan-zhan, Li Hua, Zhou Xiao-hong, Wu Kun, Yan Hui

机构信息

Experimental Animal Center, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.

出版信息

Di Yi Jun Yi Da Xue Xue Bao. 2005 May;25(5):528-30, 544.

PMID:15897125

Abstract

OBJECTIVE

To obtain soluble expression product of immunoreactive recombinant multiepitope antigen of Toxoplasma gondii from E.coli.

METHODS

The gene encoding the multiple epitopes (MEG) of Toxoplasma gondii was amplified by PCR from the original plasmid containing MEG gene and cloned into the prokaryotic soluble expression vector pET32a. After identification by enzyme digestion and sequencing, the positive recombinant plasmid pET32a-MEG was transformed into BL21(DE3), which was induced with IPTG for expression of the target antigen. The relative molecular mass, solubility and antigenicity of the expression products were analyzed by SDS-PAGE and Western blotting.

RESULTS

The recombinant expression plasmid pET32a-MEG was successfully constructed and the highly efficient expression of the antigen was achieved after IPTG induction of E.coli. Improvement of the induction condition increased the expression product which accounted for about 28% of the total bacterial protein. The target protein, with good solubility and a relative molecular mass of about 31 000, was purified by immobilized metal affinity chromatography (Ni-NTA resin) and could be well recognized by mouse and rabbit antisera derived by infection of the animals with Toxoplasma gondii B36 and RH, respectively.

CONCLUSION

The recombinant multiepitope antigen has good antigenicity and potential value in diagnosis and vaccine development of toxoplasmosis.

摘要

目的

从大肠杆菌中获得弓形虫免疫反应性重组多表位抗原的可溶性表达产物。

方法

通过PCR从含有多表位基因（MEG）的原始质粒中扩增弓形虫多表位编码基因，并克隆到原核可溶性表达载体pET32a中。经酶切鉴定和测序后，将阳性重组质粒pET32a-MEG转化至BL21(DE3)，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达目的抗原。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blotting）分析表达产物的相对分子质量、溶解性和抗原性。

结果

成功构建了重组表达质粒pET32a-MEG，IPTG诱导大肠杆菌后实现了抗原的高效表达。优化诱导条件后，表达产物增加，约占细菌总蛋白的28%。通过固定化金属亲和层析（镍-次氮基三乙酸树脂，Ni-NTA树脂）纯化得到相对分子质量约为31 000、溶解性良好的目的蛋白，该蛋白能被分别用弓形虫B36株和RH株感染动物后获得的小鼠和兔抗血清很好地识别。